Determination of Fluoride Detection and Determination of Acutely Toxic Quantities in Foods and Biological Material WILLIAM H. KING AND DOROTHY A. LUHORN Louisiana State Health Department, Division of Laboratories, N e w Orleans, La.
A detailed procedure is given for a rapid, dependable, qualitative test for detection of fluorides in fobds and biological materials. The method will detect as little as 0.04% of fluorine as fluoride even in material with high carbohydrate content. A detailed procedure is alto given for quantitative determination of fluorides in a variety of food products in the range 0.001% and up for certain carbohydrate-free foods and 0.01 % and up for pure carbohydrates.
B
ECAUSE of the frequent occurrence of cases of accidental fluoride poisoning and the numerous requests this laboratory receives for complete toxicological examination of ingested materials and organs of deceased persons, it became necessary to develop methods for rapidly and positively determining the presence or absence of acutely toxic quantities of fluorides in samples containing a high percentage of organic material. The specific nature of the glass-etching test makes this test very desirable from a qualitative standpoint. I n connection with samples containing a high percentage of organic material it is necessary t o isolate the fluoride; this is most conveniently done by ashing a portion of the sample to be tested. It has been shown (6) that in the presence of much organic matter, such as sugar, up to 80% of the fluoride content of the sample may be lost even in the presence of an alkaline fixative, such as lime suspension. Similar results have been experienc‘ed in this laboratory with samples of high sugar content, using the Briining and Quast (4, 10) method of fixing fluorides by ashing. Accordingly, the method described below was developed as a systematic procedure for testing organic material for the presence of acutely toxic amounts of fluorides. I n this method water-soluble organic material such as sugar is largely removed before ashing. The test is simple, rapid, and dependable and requires a minimhm amount of material and reagents. It has never failed to reveal the presence of 10 mg. of fluorine as sodium fluoride in 25 grams (0.04%) of a variety of foods tested in this laboratory. The test shows up unmistakably as a “frosted” circular spot about 2.5cm. (1 inch) in diameter on a microscope slide.
QUANTITATIVE DETERMINATION OF FLUORIDES
The quantitative determination of small amounts of fluoride has been the subject of considerable study by numerous investigators for over 100 years. Willard and Winter (16) introduced the distillation method for isolating fluorine, which involves constant-temperature volatilization of fluorine as hydrofluosilicic acid from aqueous solutions of sulfuric and perchloric acids. This procedure was an improvement on the volatilization methods used by Wohler (16), Offermann (11) et ul. Willard and Winter (15) also introduced the volumetric procedure of quantitative determination of fluorine based on titration with thorium nitrate, using a zirconium-alizarin mixture as indicator in alcoholic solution. Armstrong ( 8 ) titrated fluoride in an aqueous rather than alcoholic solution and Rowley and Churchill (12) ap lied the aqueous titration t o the determination of quantities o f 1 to 50 mg. of fluorine. Dahle et ul. (8) studied a “back-titration” procedure as suggested by Allen (1). This procedure has been further studied by Clifford (6) and McClure (9) and was published in an up-to-date form by the referee on waters, brine, and salt in 1942 (3). The subject of isolation of fluorine from organic material has received attention by various investigators. Preliminary distillation of fluorine from perchloric acid, in the presence of much organic material, may cause dangerous explosions. Preliminary distillation from sulfuric acid was f i s t suggested by Willard and Winter (16) and has been referred to by Wichmann and Dahle (13, 14). Direct ashing of the organic material followed by distillation of the ash has also received some attention. These efforts have been summarized by Clifford (6),but leave doubt as to the value of this method in general application to the determination of fluoride in foods and biological materials. Cox, Matuschak, Dixon, Dodds and Walker (7) state that “in agreement with Armstrong, Dafde, and McClure the value of analysis for fluorine on materials which must be ashed is questionable”. Equipped with the foregoing more or less equivocal information, the authors attempted to develop a detailed, systematic, working method for determining acutely toxic quantities of fluorine in foods and biological material. The resulting method was developed after numerous unsuccessful attempts t o arrive a t a judicious combination of detailed procedures which would isolate fluorine from various types of food and organic biological material (meat) in a form sufficiently free from interfering substances t o apply the back-titration method. At the outset it was realized that the titration procedure was sufficiently sensitive and precise to work with small samples.
QUALITATIVE TEST FOR FLUORIDES IN FOODS AND BIOLOGICAL MATERIAL
Place 25 grams of the comminuted and well-mixed sample in a 250-ml. stoppered Erlenmeyer flask, add 75 ml. of water, and mix well until any sugar or other water-soluble organic matter is in solution. Add 2 ml. of 10% calcium chloride (anhydrous), 10 ml. of 10% copper sulfate pentahydrate solution and make alkaline with sodium carbonate (10% solution), adding 15 ml. in excess. Mix well and allow t o stand in a warm lace for 1 hour. Cool to room temperature, and filter througg a fluorine-free filter paper (Whatman No. 12, 18.6-cm. folded paper is convenient), using 5 ml. of water in rinsing flask. Transfer residue and paper to a nickel evaporating dish, dry carefully, and ash a t 550” C. for 15 minutes. Crush ash and transfer to a platinum crucible. Place a clean, dry, unetched microscope slide over the crucible and add enough concentrated sulfuric acid just to cover the ash. (Previously clean the slides by immersing in cleaning solution for 0.5 hour, rinsing with distilled water, and drying in an oven.) Heat over a small flame until sulfur trioxide fumes begin to appear. Continue the heating for 15 minutes. Cool, wash the slide well with distilled water, and dry in oven. If as much as 0.04% of fluorine as fluoride was present, the glass will be distinctly etched.
To avoid loss of fluorine by direct-ashing procedures because of presence of organic material, a preliminary distillation over sulfuric acid was made. Results showed that impurities in the distillate interfered grossly with titration of fluorine. An unsuccessful attempt was made to produce a pure distillate by oxidizing the volatile organic impurities in the first distillate, concentrating in an alkaline medium, and redistilling. Inasmuch as the organic material in the first distillate is low, experiments were carried out t o determine optimum conditions for their removal by quick-ashing the first distillate after evaporating to dryness with fixative agents. These experiments resulted in the present procedure. Since samples containing a high percentage of sugar or starch produced excessive foaming in the preliminary distillation with sulfuric 4.57
INDUSTRIAL A N D ENGINEERING CHEMISTRY
458
acid, a procedure was developed largely to destroy this material with potassium permanganate. The authors feel that the workability of the back-titration method, as rewritten here, has been enhanced by use of stirring rods in the Nessler tubes. They believe that the use of a Nessler tube rack with white glass reflector and a large fluorescent titrating lamp contributes to the accuracy of the titration. Their experience shows that the titration is excellent for accurate measurement of amounts of fluorine in aliquots containing from 0.01 to 0.05 mg. after isolation of the element from interfering ions. Numerous practical details are included in both the isolation and titration procedures because it is felt that their observance is essential to the success of the method. PROCEDURE
The sample, consisting largely of organic material, is subjected to a Willard-Winter distillation using sulfuric acid, followed by evaporation of the distillate with sodium carbonate-lime water fixative and ashing for a short time in a nickel dish. The ash obtained is redistilled with perchloric acid and silver sulfate, thus providing a sufficiently pure aqueous solution of fluoride ion for accurate measurement by the back-titration procedure. REAGENTS.Sodium fluoride solution. Dissolve 2.22 grams of sodium fluoride (purity a t least 98%) in 1 liter of water (this solution contains 1 m . of fluorine per ml.). Standard sodium fuoride solution. Dilute 10 ml. of stock sodium fluoride solution to l liter (1 ml. = 0.01 mg. of fluorine). Thorium nitrate solution. Dissolve 0.25 gram of thorium nitrate dodecahydrate in l liter of water. Alizarin red indicator: Make a 0.01% water solution of sodium alizarin sulfonate. Hydrochloric acid, (1 249) (0.05 N ) . Dilute 4 ml. of hydrochloric acid to 1 liter. S o d i p hydroxide solution, 0.05 N . Potassium permanganate solution, 5%. APPARATUS.Claissen flask, capacity 125 ml. Nessler tubes, two or more long-form 50-ml. tubes matched for length, diameter, and optical similarity. Stirring rods. Out of small-diameter glass rod make stirring rods with glass rings a t bottom parallel to bottom of tube and of small enough diameter to fit into the Nessler tubes. The rods should be 5 to 7.5 cm. (2 to 3 inches) longer than the tubes, with the upper end bent at an angle, so that contents of the tube can be observed while stirring with an up and down motion. ISOLATION OF FLUORINE.Transfer 10 grams of the comminuted and well-mixed sample into the Claissen flask (rinsed with boiling lo.% sodium hydroxide to eliminate all traces of gelatinous sihca), which has been set up for a Willard and Winter (16) distillation. Willard and Winter Setup. Place the flafik on an asbestos mat with an opening large enough so that about one third of the bottom of the flask will be exposed to the flame. Close the main neck with a two-hole rubber stop er through which pass a thermometer and a capillar glass tu!, both extending within 2 to 3 mm. of the bottom o f t h e flask. Connect a small glass funnel with a stopcock to the capillary tube, so that a controped flow of water may be added during the distillation. Provlde a solid rubber stop er for the side neck and connect the flask with R water-coolelcondenser fitted with an adapter suitable for collecting the distillate in a 250-ml. Erlenmeyer flask. Add 50 ml. of sulfuric acid (1 1) and several glass beads and distill until the tem erature reaches 130" C. (use face shield during distillation). ($he amount of water in the flask is not critical, so 1n: as there is enough to cause the mixture to boil under 110 at the beginning.) Continue the distillation, holding the temperature, between 130: and 140" C. by adding water through the capillary tube until 150 ml. of distillate have been collected in that temperature range. Drain condenser water and continue the distillation until steam is evolved from the end of the condenser. Combine all distillate and transfer to a platinum or nickel evaporating dish. Neutralize with solid sodium carbonate and add 2.grams in excess. Add 10 ml. of lime water and evaporate to dryness. Ash for 5 minutes a t 550' C. Cool and completely transfer ash to a Claissen flask with water, rinsing dish with 25 ml. of 60% perchloric acid. (This operation should be carried out carefully. Add the acld through a funnel inserted ,into the side neck of the flask which has been set up for distillation.) Add ca. 0.5 gram of pure, solid silver sulfate (preventing distillation of hydrochloric acid from salt in the sample thus lowering acidity of the distillate; first suggested by McCfure, 9) and re-
distill as before, but hold the temperature of the second distillation a t 135" * 2" C. after that temperature is reached. Make up final distillate to a suitable volume-e.g., 500 m1.in a graduated flask and take a suitable aliquot for fluorine determination as follows: DETERMINATION OF ISOLATED FLUORINE. Prepare one standard and one or more sample tubes as follows: Standard Tube. Place 35 to 40 ml. of water in one of the Nessler tubes equipped with stirring rod. Add .1 ml. of the alizarin red indicator and 2 ml. of 0.05 N hydrochloric acid. Sam le Tube. Titrate an aliquot of the sample distillate with 0.05 sodium hydroxide solution, using 1 ml. of indicator. Place a suitable aliquot (0 to 0.05 mg. of fluorine) in another Nessler tube equipped with stirring rod and make up to ca. 40 ml. with water. Add enough 0.05 N hydrochloric acid to total 2 ml., including acid in the sample aliquot, if any. Add 1 ml. of the indicator, mix, and add a measured amount of thorium nitrate solution from a microburet, dropwise, until a distinct faint pink color a pears. Make up to 50-ml. mark with stirring rod resting in ut.!, Add exactly the same volume of thorium solution to the standard tube as was added to the sample tube. Back-titrate from a microburet with standard sodium fluoride solution, dropwise with stirring, until the color of the standard tube matches that of the sample tube. For find match adjust volume to that of sample tube. MI. of fluorine solution X 0.01 = mg. of fluorine in sample aliquot. Determine a reagent blank, using a sufficient amount of granulated sugar in lace of the sample. Calculate fluorine content of original sampfe, correcting for reagent blank.
d
Table 1.
Type of Sample
+
+
6.
Vol. 16, No. 7
Ground meat Granulatedeugar Flour Pea soup (concentrated) Apple jelly Cannedspnach Cola beverage Powderedegge a
Fluorine in Various Types of Foods Sodium Fi Wt. of Sample Fluoride Recovered Recovery of Ft Sample Blank" Added (Lese Blank) Grams 10 1 1
Mg. h
Mg. Fa
Ma.
%
10.00 10.00
10.00
10
0.10 0.10 0.06 0.07
9.80 9.40 9.88 9.70
98.0 94.0 98.8 97.0
1 10
0.09 0.06
10.00 10.00 10.00
10
0.10
9.92 9.92 9.43 9.32
99.2 99.2 94.3 93.2
10
0.06
10.00
10.00
Include8 reagent blank and any fluoride present in original samples.
N O T E S ON T H E M E T H O D
In case of excessive foaming or excessive charring of the sample during the distillation, such aa would be caused by samples of high carbohydrate content, use less sample. The sensitivity of the method is such that as little as 1 gram of sample can be used, provided the fluorine is evenly distributed in the sample. Samples containing more than 50y0 of sugar or starch must be decomposed with potassium permanganate solution if as much as 1 gram of sample is used. Larger samples of other material may be advantageously treated with this reagent also if excessive foaming is encountered. The reagent is used as follows: Warm sample with sulfuric acid in the Claissen flask to a temperature of 90"to 110' C. and add 5% potassium permanganate solution a few cubic centimeters at a time through a funnel in the side neck of the flask. Shake well until all the potassium permanganate has reacted, judged by di.sappearance of the pink color. Do not add an excess of otassium permanganate, as this may not only prove dangerous !ut also result in production of chlorine which would interfere in the final titration. Destroy as much organic material in this manner as is necessary t o prevent excessive foaming during distillation. It is not necessary to eliminate organic matter completely at this stage. Of all the types of foodstuffs and meat tested, only high-sugar and highstarch foods required this treatment. Results obtained by using the method on various types of food, including meat, are shown in Table I. An attempt was made to determine the source of the 0.06- t o 0.10-mg. fluorine blank obtained by applying the method to the various foods. No titration error was observed when working with the standard thorium nitrate and sodium fluoride solutions in the amount indicated in titration of the blanks.
ANALYTICAL EDITION
July, 1944
P
Taking each reagent in order, t e following blanks were obtained by Willard-Winter distillation and back-titration procedure: Blank Reagents Mg. of ‘F
+
I Perchloric acid AgxSO, I1 NatCOi I I11 Lime water (10 ml.) I 1V HrS04 I, 11. and 111 V Granulated su ar (1 ram), 50 ml. of KMnOd 11. 171, and I V
+ +
+ f,
+
0.00 0.02 0.01
0.05 0.10
These results indicate, by difference, that the sulfuric acid imparted 0.02 mg. of fluorine ta the blank. Since from 20 to 125 ml. of potassium permanganate were used with the sugar, flour, pea soup, and apple jelly, the blanks obtained with these substances indicate that the permanganate may contribute as much a8 0.05 mg. of fluorine, especially if it is assumed that pure granulated sugar contains no fluorine. The low blank obtained on spinach (which did not require use of potassium permanganate) bears this out. The high blanks obtained with ground meat and powdered eggs (0.10 mg. of fluorine), with which very little or no permanganate was used, indicate that these foods might contain 5 p.p.m. of fluorine.
459 LITERATURE CITED
(1) Allen, W. S., private communication. (2) Armstrong, W. D . , IND.ENG.CHEM., ANAL.ED.,8, 384 (1936). (3) Assoc. Official Agr. Chem., J . Assoc. Oficial Agr. Chem., 25, 101 (1942). (4) Brtlning, A., and Q u a t , H., 2. angew. Chem., 44, 656 (1931). (5) Clifford, P. A., J . Assoc. Oficial Agr. Chem., 23, 303 (1940). (6) Zbid.,24, 361 (1941). (7) Cox, G. J., Matuschak, M. C., Dixon, S. F., Dodds, M . L., and Walker, W. E., J . Dental Research, 18,486 (1939). (8) Dahle, Dan, et al., J . Assoc. Oficial Agr. Chem., 21, 459, 468 (1938). (9) McClure, F. J., IND.ENG.CHEM.,ANAL.ED.,11, 171 (1939). (10) McNslly, W. D., “Toxicology”, Chicago, Industrial Medicine, 1937. (11) Offermann, 2. angew. Chem., 3, 615 (1890). (12) Rowley, R. J., and Churchill, H. V., IND.ENQ.CHEM.,ANAL. ED.,9, 551 (1937). (13) Wichmann and Dahle, J . Assoc. Oficial Agr. Chem., 16, 620 (1933). (14) Ibid., 19, 230 (1936). (15) Willard, H. H., and Winter, 0. B., IND. ENQ.CHEM.,ANAL. ED.,5 , 7 (1933). (16) Wohler, Pogg. Ann., 48, 87 (1839).
Quantitative Determinat ion of High Molecular Weight Primary Aliphatic Amines A. W. RALSTON AND C. W. HOERR, Chemical Raearch Laboratory, Armour and Company, Chicago, 111. A simple, rapid, and accurate method for quantitative determination of primary aliphatic amines containing 1 P to 18 carbon atoms in the presence of their corresponding secondary amines i s based upon the separation of the primary amines by distillation. It can also be employed for the analysir of lower molecular weight primary amines and their salts in the absence of recondary amines.
THE
chemist working with fatty acid derivatives is frequently required t o determine the amount of high molecular weight primary amine in a sample containing primary and secondary amines or their salts. While primary aliphatic amines can readily be determined quantitatively by titration with standard hydrochloric acid solution using methyl red indicator, the analysis is complicated by the presence of secondary amines. Primary amines admixed with secondary amines have usually been separated by distillation, and then titrated with standard acid. For the high molecular weight amines, this procedure necessitates the use of vacuum distillation equipment, preferably an efficient high-vacuum still with a minimum holdup, and consequently a relatively large amount of sample is required for the analysis. In addition, this procedure requires several hours for each determination. In view of the present expansion of commercial production and development of industrial uses for aliphatic amines, a simpliEed and accurate method for their analysis is desirable. Such a method was mentioned in a recent paper (1) from this laboratory, discussing work in which it was necessary to determine the amount of amine and chloride ion in electrolyzed solutions of amine salts containing silver ions. Analysis for chloride ion in the presence of high molecular weight amines is complicated by the formation of an unfilterable colloidal heavy metal complex. Conductometric titrations are unsuccessful, owing to the fact that no sharp breaks are obtained when the conductivity of the solutions is plotted against the concentration of added electrolyte. The higher amine salts of sulfamic acid were found to be sufficiently insoluble in water to indicate their quantitative precipitation. These salts are, however, soluble
in the presence of the alkali metal or akaline earth ions which are necessarily introduced in the course of a gravimetric or conductometric analysis. The procedure which was adopted for the analysis of amine hydrochloride solutions is essentially a modification of the Kjeldahl nitrogen determination. Instead of digestion of the organic nitrogen compound with the subsequent liberation of ammonia, the primary amine is liberated by an alkali and distilled directly into an acid solution. Further investigation of the method has demonstrated its applicability to all the common salts of the primary amines as well as to mixtures of high molecular weight primary and secondary amines and their salts. METHOD AND DISCUSSION
PROCEDURE. The apparatus consists of the usual Kjeldahl setup. A Kjeldahl flask (600- or 800-ml.) is fitted with a con-
nectin bulb t o a straight condenser whose lower end is extendefby a delivery tube into a receiver. Any type of modified Kjeldahl connecting bulb may be used. To analyze for primary amine, 8 weighed portion of amine salt, or a known amount of amine salt solution, is placed in the Kjeldahl flask, the amine is liberated from its salt b addition of an excess (10% or more) of sodium (or potassium) gydroxide, and 200 to 400 ml. of water are added. The amine !i then distilled into a known amount of standard hydrochlonc acid solution. The amount of primary amine in the original sample can be determined by titrating the excess hydrochloric acid in the recbiver with standard carbonate-free alkali, using methyl red indicator. When analyzing the higher amines, it IS preferable to add a small amount of neutral ethanol to the acid in the receiver to dissolve the amine salt formed. The results of a number of determinations by this method are listed in Table I. The use of highly purified amine salts enabled accurate calculation of the amount of primary amine in the sample. Table I shows the relative amounts of sample which can be analyzed conveniently. The values for the lower amines correspond to about 0.1 to 0.6-gram samples, and for the higher amines to 0.1 to 0.2-gram samples. These amounts of the lower amines distill in less than 30 minutes. Because of the lower vapor pressures of the higher amines (8),it is convenient to
